DESCRIPTION, OVERALL (provided by applicant): The goal of this revised Program Project is to define compositions, structures, dynamics and molecular mechanisms of phosphoinositide/protein complexes that function at the interface between signaling and membrane recycling near the plasma membrane. We focus on endocytic sorting of EGFR and GLUT4 glucose transporter proteins and the recycling of GLUT4 to docking and fusion with the plasma membrane. Our proposed aims extend substantial published progress and exciting preliminary data generated during the previous grant period. These accomplishments include 1.) crystaliographic 3D structures of phosphoinositide head groups bound to FYVE and PH domains, and 3D structures of Rab regulator proteins(Project 3);2.) insight into EEA1 function in endocytosis, and EGFR sorting (Project 2);3.) definition of asymmetric microdomains of phosphoinositides in living cells, and high resolution imaging of GLUT4-containing vesicle dynamics(Project 1);the design and construction of a digital imaging microscope with unique capabilities configured for total internal reflection fluorescence (TIRF) microscopy (Core). Using TIRF, we have generated new exciting data defining novel events in EGFR sorting, and have successfully identified and quantified GLUT4 vesicle docking and fusion events. Projects 1, 2, and 3 will coordinately address the central hypothesis of the Program: that key phosphoinositide-anchored molecular complexes function in the sorting of EGFR vs. GLUT4 from early endosomes to distinct degradative vs. recycling pathways, respectively. Such complexes include one containing Rabenosyn, which binds PI(3)P through its FYVE domain, interacts with Rab5 and Rab4 through 2 other domains, and binds EHD1 through its NPF motifs;another containing EEA1 bound to PI(3)P that hypothetically directs EGFR to lysozomes;and another containing Rab11 bound to FIP2 that also associates with EHD1 and may direct exocytosis of GLUT4. TIRF and structured illumination techniques will be developed in Project 4 to achieve measurements approaching 10nm in the z-axis, and to image 3 proteins simultaneously in vesicles of living cells. Project 4 and the Core will enable Projects 1 and 2 to test key aspects of the central hypothesis. This novel technology will for the first time identify newly internalized vesicles containing EGFR or GLUT4, movements of these vesicles, docking and fusion of GLUT4 vesicles with the plasma membrane, association of these vesicles with up to three proteins simultaneously in live cells, disassembly of these proteins from the vesicles, and assembly of protein complexes onto such vesicles. Coupled with biochemical and crystaliographic methods (Project 3), the Program has the potential to define dynamic molecular mechanisms of regulated membrane retrieval and insertion at the atomic level.